Antibodies Against Synthetic Peptides Predicted from the Nucleotide Sequence of D₂ Receptor Recognize Native Dopamine Receptor Protein in Rat Striatum

Abstract

Two peptides corresponding to amino acid sequences predicted from the nucleotide sequence of the dopamine D2 receptor were synthesized. Peptide I (CGSEG-KADRPHYC) and peptide II (NNTDQNECIIY), corresponding to 24-34 and 176-185 from the NH2 terminus, respectively, were conjugated to keyhold limpet hemocyanin and injected into rabbits. Peptide I showed a greater immunogenic response than did peptide II. Both peptide antibodies exhibited high titer for the homologous antigens, but showed little or no cross-reactivity with heterogeneous peptides. Peptide I antibodies reacted with striatal membrane proteins of apparent molecular masses of 120, 90, 85, and 30 kDa on a western blot. Furthermore, the 90-kDa band was identified as denatured D2 receptor by its high affinity for the D2 selective photoaffinity probe 125I-N'-azidospiperone (125I-NAPS). Photoaffinity labeling of the 90-kDa protein by 125I-NAPS was reduced by 40% in the presence of the peptide I antibody. In addition, evidence is also presented to show the low level of 90-kDa protein in cerebellum which contains little or no D2 ligand binding sites. The antibody to peptide I inhibited the binding of [3H]YM-09151-2, a dopamine D2 receptor selective antagonist, to striatal membranes in a concentration-dependent manner; a 50% inhibition was obtained at a 1:500 dilution of the antisera with 20 pM ligand concentration. The data on the equilibrium inhibition kinetics of [3H]YM-09151-2 binding to striatal membranes were examined in the presence of antibody and showed a 25-30% decrease in Bmax (203.5 +/- 11.0 and 164.6 +/- 3.3 fmol/mg of protein in presence of preimmune and immune sera, respectively) with no change in KD.(ABSTRACT TRUNCATED AT 250 WORDS)

Parkinson's disease: Autoimmunity and neuroinflammation

“…This cytotoxic effect was further demonstrated by neuronal labelling with IgG, which correlated with neurodegeneration in PD [25]. [37,38].…”

Dopamine Inhibits Melatonin Synthesis in Photoreceptor Cells Through a D2‐Like Receptor Subtype in the Rat Retina: Biochemical and Histochemical Evidence

“…Nevertheless, the currently available immunolabeling studies do not allow the distinction between D2R and D4 R, and the recognition of a DZc/D4 subtype by the present anti-D 2 R antibody is open to question . Although the D 2AR and D 2 R share >90% homology in transmembrane domains O'Dowd, 1993), the N-terminal sequence recognized by the anti-D 2 R antibody only shares 27% with the D4 R (Farooqui et al ., 1991) .…”

Quantitative analysis of the dopamine D4 receptor in the mouse brain

“…The Kds obtained in D4RϪ/Ϫ mouse brain synaptosomes with both radioligands were similar to those obtained for D4Rϩ/ϩ mice, indicating that the affinity of the radioligands for the D2-like dopaminergic receptors was not altered for the absence of one of the members of the family. Comparisons to Kd values reported in biochemical binding assays in rat caudate putamen show that our values are an order of magnitude higher for nemonapride binding and two times higher for raclopride binding (Kohler at al., 1985;Terai et al, 1989;Farooqui et al, 1991;Vile et al, 1995). The difference might be mainly related to the species difference and to the fact that we are employing a pool homogenate of frontal cortex, caudate putamen, and hippocampus.…”